Histopathology in Mouse Metabolic Investigations

Manuel Mark1, Marius Teletin1, Cristina Antal1, Olivia Wendling1, Johan Auwerx1, Sami Heikkinen2, Konstantin Khetchoumian2, Carmen A. Argmann2, Mounzer Dgheem2

1 Institut Clinique de la Souris and Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, 2 Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 29B.4
DOI:  10.1002/0471142727.mb29b04s78
Online Posting Date:  April, 2007
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Abstract

Due to the small size of the mouse, evaluating its clinical phenotype is sometimes problematic. In contrast, mouse models are readily accessible to post‐mortem analyses at any time during the course of a disease and prior to its clinical onset. RNA, protein, and histological analyses following sacrifice represent a powerful means to identify affected cell types and molecular events underlying the altered phenotype, and therefore to understanding the signaling or metabolic pathways involved. In this unit, an overview of post‐mortem analyses is provided with a strong emphasis on the principles of routine histology, including tissue fixation, processing, embedding, and staining with hematoxylin and eosin. There are also several protocols for staining with specialized histological stains used in the metabolic field to detect intracellular lipids, intracellular lipid “ghosts”, cholesterol esters, polysaccharides, mitochondria, pathological collagen deposits, and atherosclerotic plaques.

Keywords: mouse; metabolism; histology; fixation; staining

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Fixation with Formalin
  • Basic Protocol 2: Fixation with Bouin's Solution
  • Basic Protocol 3: Fixation with Buffered Glutaraldehyde
  • Basic Protocol 4: Fixation with Buffered Paraformaldehyde
  • Basic Protocol 5: Fixation by Perfusion
  • Basic Protocol 6: Freezing Tissues in Liquid Nitrogen Vapors
  • Basic Protocol 7: Freezing Tissues in Isopentane
  • Basic Protocol 8: Hematoxylin and Eosin Staining
  • Basic Protocol 9: Oil Red O Staining for Neutral Fats
  • Basic Protocol 10: Osmium Tetroxide Staining of Unsaturated Lipids
  • Basic Protocol 11: Immunodetection of Lipid Droplet “ghosts” with Anti‐Adipophilin Antibodies
  • Basic Protocol 12: Staining for Cholesterol Esters
  • Basic Protocol 13: Periodic Acid Schiff Staining for Glycogen
  • Basic Protocol 14: Sirius Red Staining for Collagen Fibers
  • Basic Protocol 15: Succinate Dehydrogenase Staining of Mitochondria
  • Basic Protocol 16: En Face Staining of Aortas with Sudan IV
  • Reagents And Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Fixation with Formalin

  Materials
  • Mice (see )
  • 10% (v/v) neutral buffered formalin (equivalent to 4% [w/v] formaldehyde; e.g., Carlo Erba)
  • 70% ethanol
  • Plastic histo‐cassettes (e.g., Labonord)
  • Large (≥1 liter) containers with lids (e.g., Labonord)

Basic Protocol 2: Fixation with Bouin's Solution

  Materials
  • Mice (see )
  • Bouin's fixative solution (e.g., Carlo Erba)
  • 70% ethanol
  • Plastic histo‐cassettes (e.g., Labonord)
  • Large containers with lids (e.g., Labonord)
  • Rocking platform

Basic Protocol 3: Fixation with Buffered Glutaraldehyde

  Materials
  • Mice (see )
  • 2.5% (w/v) buffered glutaraldehyde in PBS (e.g., Sigma‐Aldrich), freshly prepared
  • 2‐ to 5‐ml plastic tubes with lids

Basic Protocol 4: Fixation with Buffered Paraformaldehyde

  Materials
  • Mice (see )
  • 4% (w/v) buffered paraformaldehyde (PFA) fixative (see recipe)
  • 1× phosphate‐buffered saline (PBS; e.g., Sigma‐Aldrich)
  • 70% ethanol
  • 5%, 10%, and 20% (w/v) sucrose solutions in PBS
  • Razor blades

Basic Protocol 5: Fixation by Perfusion

  Materials
  • Mice (see )
  • Anesthetic mixture (xylazine/ketamine; see recipe)
  • 70% ethanol
  • Fixative: 4% (w/v) buffered paraformaldehyde (see recipe) or 2.5% (w/v) buffered glutaraldehyde in PBS (e.g., Sigma‐Aldrich), freshly prepared
  • 1× phosphate‐buffered saline (PBS; e.g., Sigma‐Aldrich)
  • Cork board and pins
  • Dissection instruments (e.g., scissors, forceps)
  • Perfusion pump (e.g., Minipuls II, Gilson)
  • 0.8‐mm perfusion catheter (e.g., Tygon R3603 1/32 – 3/32)
  • 0.5‐mm perfusion needle (e.g., Terumo NN‐2516R)
  • 10‐ and 50‐ml plastic tubes

Basic Protocol 6: Freezing Tissues in Liquid Nitrogen Vapors

  Materials
  • Liquid nitrogen
  • Tissue freezing medium (e.g., Shandon Cryomatrix from Thermo)
  • Mice (see )
  • 1× phosphate‐buffered saline (PBS; e.g., Sigma‐Aldrich)
  • 5%, 10%, and 20% sucrose solutions in PBS
  • Dry ice
  • Styrofoam box with lid
  • Aluminum foil
  • Plastic embedding molds (e.g., Leica)
  • Dissection instruments (e.g., scissors, forceps)
  • Petri dishes
  • Filter paper

Basic Protocol 7: Freezing Tissues in Isopentane

  Materials
  • Mice (see )
  • Tissue freezing medium (e.g., Shandon Cryomatrix, Thermo)
  • Liquid nitrogen
  • Isopentane (2‐methylbutane; e.g., Sigma)
  • Dry ice
  • Dissection instruments (e.g., scissors, forceps)
  • 1.5 × 1.5–cm pieces of cork board
  • Styrofoam box with lid
  • Glass beaker (cold‐resistant)

Basic Protocol 8: Hematoxylin and Eosin Staining

  Materials
  • Histological sections on glass slides
  • Clearing agent (e.g., Histosol Plus; Shandon)
  • 95% and 100% ethanol
  • Harris hematoxylin (e.g., Merck), freshly filtered (e.g., using Rapid flow filter paper, Macherey‐Nagel)
  • Acid alcohol: 0.37% HCl in 70% ethanol
  • 0.1% (w/v) eosin Y (see recipe)
  • Permanent mounting medium (e.g., Eukitt, Labonord)
  • Staining jars or automated slide stainer
  • Hot plate at 58°C
  • 25 × 60–mm glass coverslips

Basic Protocol 9: Oil Red O Staining for Neutral Fats

  Materials
  • Histological sections on glass slides
  • 1× phosphate‐buffered saline (PBS; e.g., Sigma‐Aldrich)
  • Oil red O staining solution (see recipe)
  • Vectashield mounting medium (Vector Laboratories) containing 10 µg/ml DAPI (e.g., Roche; store in dark at 4°C)
  • Harris hematoxylin (e.g., Merck), freshly filtered (e.g., using Rapid flow filter paper, Macherey‐Nagel), optional
  • Glycerol/PBS aqueous mounting medium: 9:1 (v/v) 87% glycerol/PBS (store at 4°C), optional
  • 25 × 60–mm glass coverslips
  • Staining jars (e.g., Labonord)

Basic Protocol 10: Osmium Tetroxide Staining of Unsaturated Lipids

  Materials
  • 2 × 2–mm glutaraldehyde‐fixed tissue cubes (see protocol 3)
  • 1× phosphate‐buffered saline (PBS, e.g., Sigma‐Aldrich)
  • 4% aqueous OsO 4 (e.g., Fluka)
  • 2‐ml microcentrifuge tubes
  • Aluminum foil

Basic Protocol 11: Immunodetection of Lipid Droplet “ghosts” with Anti‐Adipophilin Antibodies

  Materials
  • 1× phosphate‐buffered saline (PBS; e.g., Sigma‐Aldrich)
  • 1× PBS with 0.05% Tween 20 (PBST)
  • Histological sections, preferably on Superfrost coated glass slides (e.g., Labonord)
  • 0.01 M sodium citrate buffer
  • Guinea pig polyclonal anti‐adipophilin antibody (Research Diagnostics)
  • Cy3‐conjugated anti–guinea pig antibody (Chemicon International)
  • Vectashield mounting medium (Vector Laboratories) containing 10 µg/ml DAPI (e.g., Roche; store in dark at 4°C)
  • Staining jars (e.g., Labonord)
  • Diamond‐tip pencil
  • 250‐ml plastic container with lid for slide holder
  • Plastic slide holder
  • Humid chamber (24 × 24 × 2–cm plastic box with opaque walls containing a water‐soaked tissue)
  • 25 × 60–mm glass coverslips
  • Fluorescence microscope equipped with DAPI and Cy3 filters
  • Additional reagents and equipment for deparaffinization and rehydration (see protocol 8)

Basic Protocol 12: Staining for Cholesterol Esters

  Materials
  • Tissue samples, fixed (see protocol 4) or frozen (see protocol 6)
  • 10% neutral buffered formalin (equivalent to 4% [w/v] formaldehyde; e.g., Carlo Erba)
  • Concentrated sulfuric acid
  • Acetic anhydride
  • Permanent mounting medium (e.g., Eukitt, Labonord)
  • Cryostat
  • Glass microscope slide
  • Glass coverslips

Basic Protocol 13: Periodic Acid Schiff Staining for Glycogen

  Materials
  • Histological sections on glass slides
  • 0.5% (w/v) aqueous periodic acid (H 5IO 6)
  • Schiff's reagent (e.g., Merck or see recipe)
  • Harris hematoxylin (e.g., Merck), freshly filtered (e.g., using Rapid flow filter paper, Macherey‐Nagel)
  • Acid alcohol: 0.37% HCl in 70% ethanol
  • Ultraclean staining jars
  • Glass coverslips
  • Automatic slide stainer (optional)
  • Additional reagents and equipment for deparaffinization, rehydration, clearing, and mounting (see protocol 8)

Basic Protocol 14: Sirius Red Staining for Collagen Fibers

  Materials
  • Histological sections on glass slides
  • 0.1% picro‐Sirius red staining solution (see recipe)
  • Acidified water: 0.5% (v/v) glacial acetic acid
  • Additional reagents and equipment for deparaffinization, rehydration, clearing, and mounting sections (see protocol 8)

Basic Protocol 15: Succinate Dehydrogenase Staining of Mitochondria

  Materials
  • Frozen histological sections on glass slides, freshly prepared
  • Succinate dehydrogenase staining solution (see recipe)
  • 1× phosphate‐buffered saline (PBS; e.g., Sigma‐Aldrich)
  • 10% (v/v) neutral buffered formalin (equivalent to 4% [w/v] formaldehyde; e.g., Carlo Erba)
  • 15% ethanol
  • Aqueous mounting medium (e.g., 90% glycerol/PBS)
  • Staining jars

Basic Protocol 16: En Face Staining of Aortas with Sudan IV

  Materials
  • Mice (see )
  • 4% (w/v) buffered paraformaldehyde (see recipe)
  • 1× phosphate‐buffered saline (PBS; e.g., Sigma‐Aldrich)
  • 70% and 80% ethanol
  • En face staining solution containing Sudan IV (see recipe)
  • Dissection instruments (e.g., scissors, forceps)
  • Dissecting microscope
  • 15‐ml tube
  • Blackened cork board
  • Insect pins (Fine Science Tools) or similar
  • Spring scissors (Fine Science Tools) or similar
  • Silicone‐coated Petri dishes (see recipe)
  • Platform rocker
  • Additional reagents and equipment for perfusion fixation (see protocol 5)
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Figures

Videos

Literature Cited

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Internet Resources
   http://www.histosearch.com
  Histosearch is a search engine that searches over 20,000 Web pages from histology‐related sites.
   http://home.primus.com.au/royellis/fix.htm
  Informational site for fixation and fixatives by A.S.‐Y. Leong.
   http://stainsfile.info/StainsFile/jindex.html
  StainsFile is a useful site with general information and resources for histology.
   http://swehsc.pharmacy.arizona.edu/exppath/resources/formaldehyde.html
  Information on formaldehyde fixatives at the Southwest Environmental Health Sciences Center (SWEHSC), University of Arizona College of Pharmacy.
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